Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-08
2003-05-27
Ponnaluri, Padmashri (Department: 1618)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S471000, C435S476000, C435S320100, C435S091500, C435S091500, C435S091500, C435S091500, C435S091500, C536S023100, C536S025320
Reexamination Certificate
active
06569623
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to genetic screening methods useful in isolating genes and identifying unknown functions of genes or unknown functional links between genes as well as in identifying molecules having gene-specific lethal properties.
BACKGROUND OF THE INVENTION
The following publications may be relevant to the invention, and are referred to in the specification by number:
1. Bender and J. R. Pringle,
Mol. Cell. Biol.
11, 1295 (1991);
2. V. Doye and E. C. Hurt,
Trends Genetics
11, 235 (1995);
3. Koshland, D. et al, Cell 40,393 (1985).
With continued progress in the Human Genome Project as well as the initiation of the Mouse/Rat Genome Projects, and the sequencing of the majority of the human cDNAs, the elucidation of gene function has become a major priority. High throughput screening methods are required in order to determine the functions of large numbers of genes in an efficient manner.
Screening methods are also required for discovering novel gene-specific drugs. In the search for such drugs, it would be advantageous to be able to elucidate the interaction between specific chemical reagents and one or more genes in a high throughput format. This is particularly relevant with respect to cancer drugs.
A synthetic or synergistic lethality screening method has previously been described in yeast cells (1,2). The essence of this screen in yeast, is the ability to identify nonallelic and nonessential mutation/s that are lethal in combination with a nonessential mutation in a gene of interest (i.e. synthetic lethality). A wild-type copy of the gene of interest, on an episomal plasmid, is introduced into cells which are null for expression of this gene. Random chemical mutagenesis of the entire yeast genome within these cells may inactivate a gene which is synthetically lethal with the gene of interest. Under these conditions, retention of the episomal plasmid, which is otherwise spontaneously lost, and expression of the gene of interest become essential for survival (i.e. survival plasmid). Plasmid loss or retention is detected by changes in colony pigmentation, due to the presence on the plasmid of a wild-type gene whose product is essential for red pigment accumulation (3). This genetic method is very powerful as it can reveal not only interactions between gene products with direct physical contacts, but also interactions along the same or parallel pathways.
A synthetic lethality screen in non-yeast eukaryotic cells in general and in human cells in particular, has not yet been described.
SUMMARY OF THE INVENTION
In an effort to expand the arsenal of genetic tools which can be applied to other eukaryotic biological systems, it has now been discovered that the concept of the yeast synthetic lethality screen can be applied to non-yeast eukaryotic cells, particularly to mammalian cells and most particularly to human cells.
An object of the present invention is to provide a screening method useful in identifying molecules having gene-specific cell-lethal properties.
A further object of the present invention is to provide a screening method useful in isolating genes and identifying unknown functions of genes or unknown functional links between genes.
In a first aspect of the invention, there is provided a method for screening a chemical library comprising a plurality of molecule types in non-yeast eukaryotic cells having a genome, in order to identify a molecule type having a gene-specific lethal property in the cell, the genome comprising a gene of interest which carries a non-lethal mutation therein.
The method comprises the following steps:
(a) transfecting a first reporter gene into the cells, and selecting clones stably expressing the reporter gene;
(b) introducing into the cells an episome comprising a functioning copy of the gene of interest, a second reporter gene, a dominant selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the episome within the cells, wherein the episome is spontaneously lost from the cells, and growing the cells in the presence of a selection compound which selects for the dominant selectable marker;
(c) selecting cell clones stably expressing the second reporter gene and the functioning copy of the gene of interest;
(d) seeding of the cells into a plurality of cell chambers and removing the selection compound;
(e) adding a molecule type of the chemical library to each of the plurality of cell chambers, measuring expression of the first and second reporter genes in the chambers, and calculating the ratio of the measured expressions, thereby indicating retention of the episome; and
(f) identifying a cell chamber in which the episome is retained, thus identifying a molecule type having a gene-specific lethal property in the cell.
In this aspect of the invention, chemical reagent induced synthetic lethality (i.e. chemical synthetic lethality) identifies biochemical inhibitors or drugs whose lethal effect is dependent on the full or partial inactivation of a specific gene (i.e. gene of interest) within its cellular milieu.
In an alternate embodiment of this aspect of the invention, there is provided a method for screening a chemical library comprising a plurality of molecule types in non-yeast eukaryotic cells having a genome, in order to identify a molecule type having a gene-specific lethal property in the cell, the genome comprising a wild-type gene of interest.
The method comprises the following steps:
(a) transfecting a first reporter gene into the cells, and selecting clones stably expressing the reporter gene;
(b) introducing into the cells an episome comprising a dominant-negative mutant of the gene of interest, a second reporter gene, a dominant selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the episome within the cells, wherein the episome is spontaneously lost from the cells, and growing the cells in the presence of a selection compound which selects for the dominant selectable marker;
(c) selecting cell clones stably expressing the second reporter gene and the dominant-negative mutant of the gene of interest;
(d) seeding of the cells into a plurality of cell chambers and removing the selection compound;
(e) adding a molecule type of the chemical library to each of the plurality of cell chambers, measuring expression of the first and second reporter genes in the chambers, and calculating the ratio of the measured expressions, thereby indicating retention of the episome; and
(f) identifying a cell chamber in which the episome is retained, thus identifying a molecule type having a gene-specific lethal property in the cell.
In a second aspect of the invention, there is provided a method for screening a collection of DNA molecules selected from the group consisting of antisense cDNA, truncated cDNA, full-length cDNA and genomic DNA, in order to identify from among them one or more modulators of gene function which are synergistically lethal to a non-yeast eukaryotic cell together with an incapacitated gene of interest, the cell having a genome which comprises the incapacitated gene of interest.
The method of this aspect of the invention comprises the following steps:
(a) transfecting a first reporter gene into the cell, and selecting clones stably expressing the reporter gene;
(b) introducing into the cells an episome comprising a functioning copy of the gene of interest, a second reporter gene, a first dominant selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the episome within the cells, wherein the episome is spontaneously lost from the cells, and growing the cells in the presence of a selection compound which selects for the first dominant selectable marker;
(c) selecting cell clones stably expressing the second reporter gene and the functioning copy of the gene of interest;
(d) incorporating each of the DNA molecules into vector vehicles containing a second dominant selectable marker gene;
(e) transfecting the cells with the vector vehicles so that each
Canaani Dan
Dafni Naomi
Dotan Iris
Oron Yoram
Simons Arnold
Eitan, Pearl, Latzer & Cohen Zedek LLP.
Ponnaluri Padmashri
Ramot University Authority for Applied Research & Industrial Dev
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